Numerous attempts have already been made to obtain antibodies having a desired specificity in high yields and with good human compatibility, in particular as therapeutic agents, the individual methods being briefly described below.
Hybridoma Antibodies.
In the 70ies, Köhler and Milstein developed a method of obtaining antibodies, i.e. the hybridoma method (Köhler and Milstein, 1975, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495-497). It first calls for an immunization of an experimental animal (usually a mouse or rat). Then, the spleen or lymph nodes are removed and the B lymphocytes contained therein in large numbers (initial stages of the antibody-producing cells) are collected. On account of “its” individual gene rearrangement, every B lymphocyte produces antibodies having only a single binding specificity. The descendents of these B lymphocytes, i.e. the B lymphocyte clones, only produce antibodies of this binding specificity.
Some of the cells obtained from the spleen of an immunized animal produce antibodies having the desired specificity. In order to be able to produce them in vitro, the B lymphocytes have to be multiplied in a cell culture. This is achieved by fusing them with myeloma cells, descendents of a plasma cell tumor. The resulting hybridoma cells show properties of both fusion partners: On the one hand, they have the immortality of cancer cells and, on the other hand, they produce the particular antibody of the B lymphocyte partner. The descendents of an individual hybridoma cell (i.e. their clone) produce antibodies having this defined specificity. They are thus also referred to as monoclonal antibodies. The method of producing hybridomas is shown in FIG. 1 by way of diagram. An advantage of the monoclonal antibodies as compared to the polyclonal antibodies is that they can be produced by the then immortal cells in a quantity which is unlimited, in principle.
Drawbacks:
In the former hybridoma method, mice are immunized and the murine B lymphocytes are then fused to a myeloma cell line. Thereafter, the thus formed hybridoma cells are propagated separately as individual cell clones and the supernatant of the individual clones is searched for antibodies having the desired specificity. Then, the identified individual clones must immediately be subcloned in a second selection run, since they are genetically instable during this period. This method is very time-consuming so that in the final analysis a maximum of several thousand hybridoma clones can be tested for the desired specificity. By means of this technique it is rather limited to establish and screen a hybridoma library. As a result, it is very difficult to automate this method. This conventional method does not permit the production of human antibodies.
Murine Strains Producing Human Hybridoma Antibodies.
Special cases are human hybridoma antibodies which can be obtained from transgenic mice, whose own immunoglobulin gene locus was replaced by parts of the human immunoglobulin gene locus (Jakobovits, 1995, Production of fully human antibodies by transgenic mice. Curr Opin Biotechnol 6, 561-566; Lonberg and Huszar, 1995, Human antibodies from transgenic mice. Int Rev Immunol. 13, 65-93; Kucherlapati et al., U.S. Pat. No. 6,114,598: Generation of xenogeneic antibodies). The human antibody genes are rearranged, pass through the class switch and are hypermutated somatically. These transgenic mice thus produce human antibodies in murine cells which (in contrast to human hybridoma cells) result in stable murine hybridomas.
Drawbacks:
Although human antibodies can be produced by means of this technique, it is as time-consuming, expensive and complex as the above discussed hybridoma technique. Little has been known about the actual quality of the generated transgenic murine strains to date. This includes questions such as: Does the interplay between humanized antibodies and other murine signals create a disturbance? What quality has the immune response of the mice? How many antibody genes function/are in the murine genome? etc. For this reason, it is not yet clear whether these “humanized mice” can meet the expectations placed on them.
Humanized Hybridoma Antibody.
A plurality of murine hybridoma antibodies which might be of therapeutic interest is already available. However, a problem with their therapeutic use is their murine origin, since proteins from a foreign species are recognized to be foreign by the human immune system. This also applies to murine antibodies. What is called the “HAMA” immune response (human anti-murine antibodies) occurs. These antibodies formed by the human immune system within some days usually neutralize the therapeutically used murine antibody, thus rendering it ineffective. Also, a repeated therapy is only possible to a very limited extent (Courtenyl-Luck et al., 1986, Development of Primary and Secondary Immune Responses to Mouse Monoclonal Antibodies Used in the Diagnosis and Therapy of Malignant Neoplasms. Cancer Res. 46, 6489-6493; Lamers et al., 1995, Inhibition of bispecific monoclonal antibody (bsAb) targeted cytolsis by human anti mouse antibodies in ovarian carcinoma patients treated with bsAb targeted activated T lymphocytes. Int J Cancer 60, 450-457).
The large majority of the HAMA antibodies is directed against the constant antibody part and this is why the production of antibody chimeras has been favored. The latter contain a variable mouse antibody domain, followed by the constant antibody domains from humans. For this purpose, a human antibody gene is initially inserted in a cloning vector (Wright et al., 1992, Genetically engineered Antibodies: Progress and Prospects. Critical Rev Immunol. 12, 125 168). The individual antibody domains form compact folding units which are interconnected by a peptide strand. The possibility of a disturbance of the antibody function is the least when whole antibody domains are exchanged. By means of the PCR invention it is possible, without any problems, to produce chimeric cDNAs since cloning down to the base is substantially simplified by this method. The resulting chimeric antibodies still bind specifically to the antigen. Yet the HAMA response is markedly reduced.
However, another fact is more important than the HAMA response: Since the constant domains are now derived from humans, these chimeric antibodies are also markedly better for activating some helper functions of the human immune system, such as antibody dependent cellular cytotoxicity (ADCC) or complement activation. This is another reason why some of these humanized antibodies are already in clinical use (McLaughlin et al., 1998, Clinical status and optimal use of Rituximab for B-cell lymphomas. Oncology 12, 1763-1777).
Drawbacks:
The humanization of already existing hybridoma antibodies is also very difficult and time-consuming. The stability of the thus produced hybridomas often creates a problem: The cells mutate or they secrete only some antibodies into the medium.
Humanization by Homologous Recombination.
U.S. Pat. No. 5,202,238 describes a method by which monoclonal murine antibodies can be humanized. This method focuses on what is called “homologous recombination”. Here, human sequences flanked by suitable genomic murine sequences are recombined in the active antibody site at the proper locations. The major advantage of this method is that the signals, optimized with respect to good antibody production, of the antibody site are largely maintained (Yarnold and Fell, 1994, Chimerization of antitumor antibodies via homologous recombination conversion vectors. Cancer Research 54, 506-512; Fell et al., 1989, Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting. PNAS 86, 8507-8511).
Drawbacks:
Similar to the generation of hybridomas this method calls for a lot of work in order to isolate a single humanized hybridoma. Thousands of clones have to be cultured and analyzed separately for this. Another drawback results from the employed selection marker: It obviously causes a large number of revertants (the human antibody locus recombined thereinto is again excised in the reverse reaction) and/or an impairment of the expression level (Baker et al., 1994, J. Immunological Methods 168, 25-32). In addition, the inserted resistance genes prevent the surface presentation of the antibodies when they are inserted in the intron between the CH3 exon and the M1 exon of an IgG.
Recombinant Antibodies.
In the last few years, a possibility based on genetic-engineering methods for the production of antibody fragments was opened up by the construction of recombinant antibodies (Breitling and Dübel, 1997, “Rekombinante Antikörper” [recombinant antibodies], Spektrum-Verlag ISBN 3-8274-0029-5). Here, the antibodies are no longer produced in an experimental animal (or in a human organism) but in vitro in bacteria or a cell culture and the focus is laid on the antigen-binding part of the antibody. Usually the rest of the antibody molecule is dispensed with to the advantage of a greater yield. Of course, these fragments can no longer convey all the functions of a naturally produced antibody. However, they can be fused in a comparatively simple way with enzymes or other antibodies. These recombinant antibodies are thus given completely new properties. The term “recombinant antibody” has become established for an antibody fragment produced in vitro by means of genetic engineering and otherwise defined exclusively via its antigen specificity.
Drawbacks:
Recombinant antibodies lack the constant antibody portion and thus the effector functions essential for many therapy approaches. For this reason, newly discovered “antibody heads” are “grafted” onto eukaryotic expression vectors for many applications, i.e. the above described labor-intensive method is used. Along with the resulting experimental work, a poor expression may result since the bacterial system prefers codons differing from those preferred in the eukaryotic systems. Another drawback results from the properties of the “single chain” antibodies usually used (svFv): They are usually rather unstable and aggregate readily. In addition, many variable domains are obviously attacked by E. coli proteases. Thus, the published complexities of the scFv antibody libraries (up to 1011) also have to be taken with caution. In addition to the just described problems, these libraries obviously also still contain a large number of cloning artifacts. This in turn means that the selected clones represent almost exclusively artifacts after 4 selection runs at the latest. Another drawback is the fact that usually more than one selection run is required to obtain the desired antibody. This is because per phage only about 0.1 scFv antibodies are presented on the phage surface. It is very likely that this value varies widely, depending on the presented antibody. Another drawback is that the identities of the individual clones (i.e. does the selected clone actually produce an antibody?) have to be checked in a rather time-consuming and costly manner.
Presentation of Antibodies on the Surface of Hybridoma Cells.
This technique is based on the above described hybridoma technique. In contrast thereto, however, the latter uses (and produces) a stable myeloma cell line which anchors large amounts of an antibody binding protein (e.g. protein G) on the cell surface (Breitling et al., 1999, Selektion von monoklonalen Antikörpern [selection of monoclonal antibodies]. DE 199 00 635 A1. PCT-Application under number PCT DE00/00079). This serves for avoiding a major part of the work which results from the cloning and subcloning of the monoclonal hybridomas. The desired antibody specificities can be isolated in a FACS sorter or with magnetobeads from a pool of hybridomas instead, since the produced antibodies are anchored to the described antibody binding protein on the cell surface as a result of the bond.
Drawbacks:
This technique prevents only part of the time-consuming work required for selecting individual antibody specificities. Mice still have to be immunized and the murine B lymphocytes then have to be fused with a myeloma cell line. This is done with relatively poor efficiency: Only 100-500 different hybridomas are usually generated per fusion and mouse. The thus formed hybridoma cells also have an undesired high variability as regards the number of presented antibodies, which strongly impairs the selection in the FACS sorter. In addition, a cross-talk between different antibody specificities results because of the non-covalent antibody anchorage on the surface. As a result, the different hybridoma cells do not only present “their” specific antibody on the surface but also other antibody specificities which are released into the medium by other hybridoma cells. This method is also time-consuming so that in the final analysis only a maximum of several ten thousand different hybridomas can be generated (and can then be tested for the desired specificity). Thus, the establishment and the screening of a hybridoma library by means of this technique are limited. This also applies to the automation of this method. Moreover, this technique also fails to enable the production of human antibodies.
Cassette Exchange by Means of Specific Recombination.
There are meanwhile a number of methods enabling a DNA site-specific recombination within a eukaryotic cell (see e.g. Sauer, U.S. Pat. No. 4,959,317: Site-specific recombination of DNA in eukaryotic cells: Leboulch et al., U.S. Pat. No. 5,928,914: “Methods and compositions for transforming cells; Feng et al., 1999, Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J. Mol. Biol. 292, 779-785). All of these methods use recombinase (e.g. Flp, Cre, Int, etc.) which recognizes specific DNA sequences and recombines them with other DNA sequences. Characteristics of these methods are the often rather high efficiencies of the specific recombination events which can be achieved in vitro but also in vivo by means of these methods. These methods are applied, e.g. as cloning aids (exchange of DNA cassettes in vitro) but also in vivo for a recombination in living bacteria, in living eukaryotic cells and even in transgenic mice.
Drawbacks:
The cassette exchange of antibody genes in eukaryotic cells in combination with the surface expression and subsequent selection of monoclonal antibodies has not yet been described.